Carotenoids are pigments that are ubiquitous throughout nature and synthesized by all photosynthetic organisms, and in some heterotrophic growing bacteria and fungi. Carotenoids provide color for flowers, vegetables, insects, fish, and birds. Colors range from yellow to red with variations of brown and purple. As precursors of vitamin A, carotenoids are fundamental components in our diet and they play an important role in human health. Industrial uses of carotenoids include pharmaceuticals, food supplements, animal feed additives, and colorants in cosmetics, to mention a few. Because animals are unable to synthesize carotenoids de novo, they must obtain them by dietary means. Thus, manipulation of carotenoid production and composition in bacteria can provide new or improved sources for carotenoids.
Carotenoids come in many different forms and chemical structures. Most naturally occurring carotenoids are hydrophobic tetraterpenoids containing a C40 methyl-branched hydrocarbon backbone derived from successive condensation of eight C5 isoprene units (IPP). In addition, novel carotenoids with longer or shorter backbones occur in some species of nonphotosynthetic bacteria. Carotenoids may be acyclic, monocyclic, or bicyclic depending on whether the ends of the hydrocarbon backbones have been cyclized to yield aliphatic or cyclic ring structures (G. Armstrong, (1999) In Comprehensive Natural Products Chemistry, Elsevier Press, volume 2, pp 321–352).
Carotenoid biosynthesis starts with the isoprenoid pathway to generate the C5 isoprene unit, isopentenyl pyrophosphate (IPP). IPP is then condensed with its isomer dimethylallyl pyrophosphate (DMAPP) to generate the C10 geranyl pyrophosphate (GPP) which is then elongated to form the C15 farnesyl pyrophosphate (FPP). FPP synthesis is common in both carotenogenic and non-carotenogenic bacteria. Additional enzymes in the carotenoid pathway are able to then generate carotenoid pigments from the FPP precursor, segregating into two categories: (i) carotene backbone synthesis enzymes and (ii) subsequent modification enzymes. The backbone synthesis enzymes include geranyl geranyl pyrophosphate synthase, phytoene synthase, phytoene dehydrogenase and lycopene cyclase, etc. The modification enzymes include ketolases, hydroxylases, dehydratases, glycosylases, etc.
It is known that β-carotene can be converted to isorenieratene, an aromatic carotenoid, by a CrtU carotene desaturase. The crtU gene, encoding the carotene desaturase, has been identified in a few actinomycetes including Streptomyces, Mycobacterium and Brevibacterium (Krugel et al., Biochimica et Biophysica Acta, 1439: 57–64 (1999); Krubasik and Sandmann, Mol Gen Genet 263: 423–432 (2000); and Viveiros et al., FEMS Microbiol Lett, 187: 95–101 (2000)). Another aryl-carotene, chlorobactene, was reported in photosynthetic green bacteria (Liaaen-Jensen et al., Acta Chem. Scand 18: 1703–1718 (1964); Takaichi et al., Arch Microbiol, 168: 270–276 (1997)). Recent genomic sequencing of Chlorobium tepidum identified a putative carotene desaturase gene (Eisen et al., PNAS USA, 99: 9509–9514 (2002), which might be responsible for the synthesis of the native chlorobactene and derivatives. However, function of the putative carotene desaturase gene from Chlorobium has not yet been determined. It is likely that the CrtU from actinomycetes might also act on other substrates in addition to β-carotene to produce a variety of aryl-carotenoids, such as converting γ-carotene to chlorobactene.
Schumann et al. (Mol Gen Genet, 252: 658–666 (1996)) reported difficulty in attempting to express crtU in heterologous hosts. However, Lee et al. (Chem Biol 10(5): 453–462 (2003)) recently reported successful expression of the Brevibacterium linens crtU (DSMZ 20426) in E. coli using a pUC-derived expression vector. Lee et al. were able to detect the production of isorenieratene (in cells engineered to produce β-carotene) and didehydro-β-θ-carotene (in cells engineered to produce torulene). Lee et al. did not report the levels of aromatic carotenoids produced. It is likely the level was low since a low copy number pACYC-base plasmid was used to produce β-carotene precursor in a non-engineered E. coli host. Production of commercially-significant amounts of aryl carotenoids has not been reported in the literature.
Expressing genes from gram positive bacteria (with high G+C content) in E. coli is known to be often difficult. Low yields of protein in heterologous expression systems can been attributed to differences in codon usage. Difficulties in expressing heterologous genes in a host strain are generally due to an extremely rare codon used by host strain and correlates with low levels of its corresponding tRNA.
The inability to adequately express CrtU carotene desaturases in a gram-negative host for production of aryl carotenoids at commercially-useful levels presents a significant hurdle to the synthesis of a variety of aryl-carotenoids by genetic engineering. Furthermore, natural aryl-carotenoids are always present as mixtures of the aryl-carotenoid with their precursors or derivatives (Kohl et al., Phytochemistry, 22: 207–213 (1983); Takaichi et al., supra). Production of a pure aryl-carotenoid requires the ability to efficiently express the carotene desaturase in an industrially-useful heterologous host, such as E. coli. 
The problem to be solved is to express a functional carotene desaturase (crtU) gene for the production of aryl-carotenoids in a gram-negative production host at commercially-significant concentrations. Applicants have solved the stated problem by isolating the crtU gene from Brevibacterium linens and expressing an optimized version of this gene in an Escherichia coli strain engineered to produce high levels of carotenoids.